Hi all,
I extracted total RNA from the liverwort Marchantia polymorpha using QIAGEN's RNeasy Plant Mini Kit and digested half of what I obtained with Ambion's TURBO DNase. When I ran it on Agilent's TapeStation to check for integrity, I obtained something unusual, i.e. there was a clear band shift between the digested RNA versus the undigested RNA coming from the same extraction. The shift was the same in all technical replicates and I think it's not due to secondary structure, because I denatured it for 3 minutes at 72 degrees before running on the TapeStation. Any idea what might be causing it?
Dear Giulia, the shift was upper side or lower side?
I would suggest first check the concentration of your RNA before subjecting it to Tapestation. If it is too concentrated I would dilute it and retest. It will also depend on what is your downstream application for your RNA. If you are really using it for NGS I would be worried (most) the RIN No. results rather than structures.
Kind regards
Solomon
Dear Solomon,
thanks for your answer, the shift is towards the lower side of the gel. When i compare the electropherograms, I do similar profiles and my RIN values are good. I'm going to use the RNA for qRT-PCR so I need to be sure of its integrity, I am thinking of carrying out a test run using a housekeeping gene and the same concentration of RNA to check if the Ct values differ significantly among independent extractions with the same method.
I will also check again the concentration of the RNA to make sure I'm not overloading the tape.
All the best,
Giulia
Great, yes I think a control will work better to rule out anything. You want to ensure also your TP reagents thaws properly for a better fidelity.
Best wishes
Solomon
- Badges
- Science topic
- Similar topics
- Mathematics